Vaccinia virus and more recently other poxviruses have been used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus (Piccini et al., 1987).
Specifically, the recombinant poxviruses are constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of the vaccinia virus described in U.S. Pat. No. 4,603,112, the disclosure of which patent is incorporated herein by reference.
First, the DNA gene sequence to be inserted into the virus, particularly an open reading frame from a non-pox source, is placed into an E. coli plasmid construct into which DNA homologous to a section of DNA of the poxvirus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA containing a nonessential locus. The resulting plasmid construct is then amplified by growth within E. coli bacteria (Clewell, 1972) and isolated (Clewell et al., 1969; Sambrook et al., 1989).
Second, the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively gives a poxvirus modified by the presence, in a nonessential region of its genome, of foreign DNA sequences. The term "foreign" DNA designates exogenous DNA, particularly DNA from a non-pox source, that codes for gene products not ordinarily produced by the genome into which the exogenous DNA is placed.
Genetic recombination is in general the exchange of homologous sections of DNA between two strands of DNA. In certain viruses RNA may replace DNA. Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.
Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs. A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.
However, recombination can also take place between sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable by the presence of that genetic marker or gene in the recombinant viral genome.
Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessential region of the virus in order that the modified virus remain viable. The second condition for expression of inserted DNA is the presence of a promoter in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed.
The technology of generating vaccinia virus recombinants has recently been extended to other members of the poxvirus family which have a more restricted host range. The avipox virus, fowlpox, has been engineered as a recombinant virus. This recombinant virus is described in PCT Publication No. WO89/03429.
Fowlpox virus (FPV) has advantageously been engineered as a vector expressing antigens from poultry pathogens. The hemagglutinin protein of a virulent avian influenza virus was expressed in an FPV recombinant (Taylor et al., 1988). After inoculation of the recombinant into chickens and turkeys, an immune response was induced which was protective against either a homologous or heterologous virulent influenza virus challenge (Taylor et al., 1988). In addition, the surface glycoproteins (fusion and hemagglutinin) of a virulent strain of Newcastle Disease Virus have been expressed in an FPV vector and shown to induce a protective immune response (Taylor et al., 1990; Edbauer et al., 1990).
FPV is the prototypic virus of the Avipox genus of the Poxvirus family. The virus causes an economically important disease of poultry which has been well controlled since the 1920's by the use of live attenuated vaccines. Replication of the avipox viruses is limited to avian species (Matthews, 1982) and there are no reports in the literature of the virus causing a productive infection in any non-avian species including man. This host restriction provides an inherent safety barrier to transmission of the virus to other species and makes use of FPV as a vaccine vector in poultry an attractive proposition.
Infectious bursal disease, also known as Gumboro's disease, manifests itself in two ways. In chickens older than three weeks, infectious bursal disease virus (IBDV) can cause impaired growth and mortality losses of up to 20% (Lukert and Hitchner, 1984). In younger birds, the disease is subclinical but is evident as microscopic lesions in the bursa of Fabricius (Winterfield et al., 1972). This results in prolonged and severe immunosuppression which causes increased susceptibility to disease and interferes with vaccination programs against other disease agents (Allan et al., 1972). Characteristics of the disease have been reviewed in Lukert and Saif (1991) and will be summarized briefly here.
The cloacal bursa appears to be the primary target organ of the virus and birds surgically bursectomized at 4 weeks have been shown to survive a lethal IBDV challenge without clinical manifestations (Kaufer and Weis, 1980). The age of bursectomy is however, critical. Schat et al., (1981) performed embryonal bursectomy and then challenged with IBDV at 2 and 6 weeks of age. Birds developed typical hemorrhagic lesions, were clinically ill and showed some mortality. The target cells are actively dividing B lymphocytes (Muller, 1986; Burkhardt and Muller, 1987). Muller (1986) demonstrated that IBDV will replicate preferentially in lymphoid cells from the bursa and poorly in lymphoid cells from other organs. It has been proposed that clinical signs of IBDV infection may result from immune complex formation (Ley et al., 1979; Skeeles et al., 1979). Muller (1986) however, demonstrated that the preferential replication in the lymphoid cells of the bursa is not related to the presence of surface immunoglobulins.
Two serotypes of IBDV, designated 1 and 2 have been demonstrated (McFerran et al., 1980; Jackwood et al., 1984; McNulty and Saif, 1988). Virulent serotypes have been shown in Group 1. No disease has been associated with group 2 viruses. In addition, considerable antigenic variation has been documented within serotypes (Lukert and Saif, 1991).
The causative agent, IBDV, has been classified as a Birnavirus (Brown et al., 1986). The biochemistry and replication of IBDV has been reviewed in Kibenge et al., (1988). Birnaviruses are small non-enveloped animal viruses having two segments of double-stranded RNA. The smaller genomic segment (segment B) of IBDV encodes a single polypeptide of 90k designated VP1. This protein is a minor internal component of the virion and is presumed to be the viral RNA polymerase (Hudson et al., 1986; Nagy et al., 1987; Spies et al., 1987). The larger genomic segment (segment A) encodes 5 polypeptides with the following designations and approximate molecular weights 52k (VPX), 41k (VP2), 32k (VP3), 28k (VP4) and 16k (Azad et al., 1985). The identity and presence of the 16K polypeptide has not been confirmed (Kibenge et al., 1988). VP2, VP3 and VP4 arise by co-translational proteolytic cleavage of precursor polyproteins. The protein VP4 is thought to be a viral protease (Hudson et al., 1986) responsible for cleavage between VPX and VP4 (Duncan et al., 1987) and between VP4 and VP3 (Azad et al., 1987; Jagadish et al., 1988).
Protein VP2 is the most abundant protein of the viral capsid making up 51% of serotype I IBDV proteins (Dobos et al., 1979). VP2 is only found in mature vital particles and is not seen in IBDV infected cells (Becht et al., 1988). VP2 is thought to be a specific cleavage product of a VPX precursor. Peptide mapping has shown that VPX and VP2 of IBDV strain CU-1 have similar amino acid sequences (Muller and Becht, 1982; Dobos, 979). In addition both VPX and VP2 react with the same monoclonal antibody on Western blots (Fahey et al., 1985b; Becht et al., 1988). It has recently been demonstrated that a conformational dependent neutralizing epitope exists on VP2 (Azad et al., 1987; Fahey et al., 1989) and a conformation independent neutralizing epitope exists on VP3 (Fahey et al., 1985 a,b). Antibodies to these epitopes were found to passively protect chickens (Fahey et al., 1985b; Azad et al., 1987; Fahey et al. 1989). Becht et al., (1988) and Snyder et al., (1988) indicated that neutralizing monoclonal antibodies to VP2 differentiated between serotypes 1 and 2 in cross-neutralization tests. However, Becht et al., (1988) also indicated that monoclonal antibodies to VP3 recognized a group-specific antigen from both serotypes which was not associated with neutralizing activity or protection. These studies may indicate the existence of multiple epitopes at least on VP2 and perhaps on VP3.
In a recent publication, Macreadie et al., (1990) demonstrated the expression of VP2 in a yeast vector. The size of the expressed protein was consistent with that of an authentic VP2. Centrifugation and gel filtration studies indicated that the VP2 expressed in yeast was in a high molecular weight aggregated form. Chickens inoculated with a crude extract of the yeast expressed VP2 developed an immune response as demonstrated by ELISA test and virus neutralization tests. One day old chickens were then inoculated with anti-sera from chickens previously inoculated with yeast expressed VP2. These chickens were passively protected against IBDV challenge as evidenced by lack of IBDV antigen in the bursa (Macreadie et al., 1990).
Current vaccination strategies against IBDV include both live and killed vaccines. Antibody transmitted from the hen via the yolk of the egg can protect chickens against early infections with IBDV. Therefore, use of killed vaccines in oil emulsions to stimulate high levels of maternal antibody is extensive in the field (Lukert and Saif, 1991). Studies by Lucio and Hitchner (1979) and Baxendale and Lutticken (1981) indicated that oil emulsion IBDV vaccines can stimulate adequate maternal immunity to protect chickens for 4-6 weeks. In contrast progeny from breeders vaccinated with live vaccines are protected for only 1-3 weeks after hatching (Lukert and Saif, 1991).
Determination of when maternal antibody has waned, and thus when antibody levels can be boosted by active immunization is problematical. It is therefore common practice to vaccinate all chicks against IBD with a live vaccine during the first 3 weeks of life (Winterfield et al., 1980). Inactivated vaccines are ineffective in inducing active immunity in chicks with maternal antibody. Presently available live vaccines consist of strains of intermediate virulence or highly attenuated strains, as well as some cell culture adapted variant strains. While intermediate strains can break through maternal antibody titers of approximately 1:250 (Lukert and Saif, 1991), the strains vary in virulence and can induce bursal atrophy and immunosuppression in day old and 3 week old SPF chickens (Lukert and Mazariegos, 1985).
Given the limitations of current vaccination strategies, it can be appreciated that provision of an IBDV recombinant poxvirus, and of vaccines which provide protective immunity against IBDV infections, would be a highly desirable advance over the current state of technology.